CN117106015B - 一种椰子源活性肽及其应用 - Google Patents
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Abstract
本发明涉及生物活性肽技术领域,尤其涉及一种椰子源活性肽及其应用。一种椰子源活性肽在制备具有酪氨酸酶抑制功效的制品中的应用,所述的椰子源活性肽的氨基酸序列为DGE。优点在于:活性肽Asp‑Gly‑Glu能够与酪氨酸酶进行稳定有效的结合、抑制酪氨酸酶的活性,从而抑制黑色素的生成,有助于改善人面部的色素沉着。可应用于制备具有美白功效的化妆品及功能性食品,安全无毒,易消化吸收。
Description
技术领域
本发明涉及生物活性肽技术领域,尤其涉及一种椰子源活性肽及其应用。
背景技术
酪氨酸酶(EC 1.14.18.1)是一种含铜的多功能酶,也被称为多酚氧化酶。它在许多生物过程中发挥作用,包括抗氧化、免疫调节和药物代谢。酪氨酸酶催化酪氨酸的氧化聚合形成多巴醌,这是黑色素合成的必要步骤之一。酪氨酸酶作为黑色素合成的关键限速酶,催化L-酪氨酸羟基化为L-多巴和多巴氧化为多巴醌。这些化学反应是合成黑色素的必要步骤,黑色素随后通过一系列自发反应产生,包括邻醌和其他中间物的氧化、聚合和重排。黑色素的过度产生可能导致人类皮肤的色素不均匀,并形成色素紊乱。此外,过度的酪氨酸酶活性可能导致氧化压力,引起细胞损伤、炎症和癌症等疾病。研究表明,酪氨酸酶的过度表达和过度激活也可能与其他一些疾病的发展和进展有关。因此,抑制酪氨酸酶的催化活性是预防和改善酪氨酸酶相关问题的主要方法之一。
由于酪氨酸酶在黑色素的生成和褐变过程中起到关键作用,从植物、细菌和真菌的代谢物中分离得到的天然来源的抗酪氨酸酶活性物质越来越受到关注,因其毒性较小并有更好的生物利用度,特别适用于食品、化妆品和药物领域的应用。椰子作为我国南方广泛存在的水果,研究表明,椰子蛋白具有广泛的生理活性,如抗氧化活性、抗菌活性、降血压活性等。其中,椰子中最主要的蛋白质是椰子乳球蛋白,不仅含量高,而且营养价值高,为人体提供赖氨酸等必需氨基酸。但目前未有公开报道显示椰子中具有酪氨酸酶抑制作用的活性肽。
发明内容
本发明为解决上述问题,提供一种椰子源活性肽及其应用。
本发明目的在于提供一种椰子源活性肽在制备具有酪氨酸酶抑制功效的制品中的应用。
优选的,椰子源活性肽的氨基酸序列为DGE。
优选的,制品为功能性食品、美白药物或美白化妆品。
与现有技术相比,本发明能够取得如下有益效果:
本发明椰子源活性肽DGE能够有效抑制酪氨酸酶活性,通过抑制酪氨酸酶活性抑制黑色素的形成,具有皮肤美白功效。与其它美白类产品相比,多肽对皮肤无害且可以经口服用,快速被人体消化吸收到达作用靶点,更好地发挥美白功效。
附图说明
图1是根据本发明实施例提供的活性肽DGE与酪氨酸酶的2D相互作用结果图。
具体实施方式
在下文中,将参考附图描述本发明的实施例。在下面的描述中,相同的模块使用相同的附图标记表示。在相同的附图标记的情况下,它们的名称和功能也相同。因此,将不重复其详细描述。
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及具体实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,而不构成对本发明的限制。
实施例1
一、活性肽的计算机虚拟筛选
借助NCBI(https://www.ncbi.nlm.nih.gov/)数据库获得椰子11s球蛋白的氨基酸序列,并使用在线酶切工具ExPASy PeptideCutter对获得的序列进行虚拟酶切。通过在线程序Peptide property calculator和ToxinPred预测多肽的水溶性和毒性。将筛选的无毒并且水溶性良好的多肽定义为配体,从Protein Data Bank(PDB)数据库获取酪氨酸酶的3D结构(PDB ID:2Y9X)并将其作为受体蛋白,通过Discovery Studio(DS)软件进行分子对接筛选能与酪氨酸酶活性位点紧密结合的肽,初步确定潜在的具有酪氨酸酶抑制作用的活性肽AENG、NSEK、GETV、DGE。具体操作如下:
借助NCBI(https://www.ncbi.nlm.nih.gov/)数据库获得11s球蛋白(Accession:AKS26848.1)的氨基酸序列,并使用在线酶切工具ExPASy PeptideCutter(http://web.expasy.org/peptide_cutter/)利用胰蛋白酶(EC 3.4.21.4)、胃蛋白酶(EC3.4.23.1)和胰凝乳蛋白酶(EC 3.4.21.1)对获得的序列进行虚拟酶切,得到93条肽段。利用在线程序Peptide property calculator和ToxinPred预测多肽的水溶性和毒性,筛选得到水溶性好且无毒的67个肽。从Protein Data Bank(PDB)数据库获取酪氨酸酶的3D结构(PDB ID:2Y9X)并将其作为受体蛋白,并在DS程序的受体-配体相互作用模块定义活性位点,将筛选的无毒并且水溶性良好的多肽定义为配体,并进行能量最小化处理。采用DS程序中的CDOCKER模块来筛选能与酪氨酸酶活性位点有效结合的活性肽,其对接坐标为x:-10.0,y:-28.8,z:-43.6,半径为较低的CDOCKER-ENERGY值意味着活性肽与酪氨酸酶的结合越紧密。对接结果表明,32个肽可以与酪氨酸酶活性位点结合。将已知酪氨酸酶抑制肽DGL(-112.13kcal/mol)和FPY(-40.183kcal/mol)作为阳性对照。如表1所示,筛选得到四种最具有潜在酪氨酸酶抑制活性的多肽AENG、NSEK、GETV、DGE。其CDCODER-ENERGY值分别为-118.183、124.56、111.026、135.508。且对接能量值均低于DGL和FPY。
表1潜在蛋清源酪氨酸抑制肽的水溶性、毒性和分子对接结果
二、体外酪氨酸酶抑制活性测定
通过分光光度计法分别对活性肽AENG、NSEK、GETV、DGE的酪氨酸酶的抑制活性进行测定。取80μL不同浓度的肽溶液,加入40μL(500U/mL)酪氨酸酶溶液,在25℃条件下孵育5min。随后加入80μL的L-多巴溶液(0.5mM)启动反应,在25℃条件下反应5min,反应结束后迅速测定反应液在475nm处的吸光度值。每次平行测量重复三次。利用下列公式计算酪氨酸酶抑制率:
酪氨酸酶抑制率(%)=[(A-B)-(C-D)]×100/(A-B)
其中A是不含抑制剂反应液的吸光度值,B是不含抑制剂和酶反应液的吸光度值,C是含有L-多巴反应液的吸光度值,D是不含有酶反应液的吸光度值。IC50定义为在测定条件下抑制50%酪氨酸酶活性的抑制剂浓度。
结果表明,肽DGE可有效抑制酪氨酸酶活性,其IC50值3.06mg/mL。而肽AENG、NSEK、GETVD未表现出酪氨酸酶抑制活性。
三、确定具有酪氨酸酶抑制作用的活性肽
椰子蛋白源酪氨酸酶抑制肽DGE与酪氨酸酶(2y9x)对接的2D可视化结果如图1所示,结果表明活性肽与酪氨酸酶残基His61,His296,Asn260,Arg268结合,氢键和疏水相互作用是肽与酪氨酸酶结合的主要作用力。
DGE的氨基酸序列为Asp-Gly-Glu。
实施例2
通过计算机虚拟筛选,高效地从椰子乳球蛋白中鉴定到新型的酪氨酸酶抑制肽DGE;在实际生产中,酪氨酸酶抑制肽DGE可通过固相合成法或定向生物酶解及纯化的方法获得,制备成颗粒状、粉末状或溶于水的液体添加到功能食品中,有助于改善人面部的色素沉着。也可将活性肽添加到常规洗面奶、面膜及面霜中制备美白类化妆品。
应该理解,可以使用上面所示的各种形式的流程,重新排序、增加或删除步骤。例如,本发明公开中记载的各步骤可以并行地执行也可以顺序地执行也可以不同的次序执行,只要能够实现本发明公开的技术方案所期望的结果,本文在此不进行限制。
上述具体实施方式,并不构成对本发明保护范围的限制。本领域技术人员应该明白的是,根据设计要求和其他因素,可以进行各种修改、组合、子组合和替代。任何在本发明的精神和原则之内所作的修改、等同替换和改进等,均应包含在本发明保护范围之内。
Claims (1)
1.一种椰子源活性肽在制备具有酪氨酸酶抑制功效的美白药物或美白化妆品中的应用,其特征在于,所述的椰子源活性肽的氨基酸序列为 DGE。
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